Dissertations / Theses on the topic 'In-situ hybridisation'
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1
Princivalle, Alessandra Patrizia. "Studies of GABAb receptors in epilepsy." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.289300.
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The binding of a novel GABAs receptor radioligand eH]-CGP62349 to human hippocampal control and epileptic sections was investigated using quantitative receptor autoradiography. Kinetic analyses performed on rat brain sections, to conserve the use of human tissues, demonstrate that eH]-CGP62349 associated rapidly. The same radioligand dissociated rapidly initially, then very slowly. Utilising human hippocampus it was shown that CH]-CGP62349 bound with high affinity (O.SnM) to human control hippocampal sections. The kinetics of GABAs receptor in human hippocampus using the novel compound confirmed previous studies performed in rat membrane. The localisation of GABAB receptors in human hippocampal control partially supported former studies using agonist ligands such as CH]-GABA and eH]-baclofen, despite differences have been noticed. Hippocampal slices from surgical resected specimens obtained from hippocampal sclerotic (HS)/temporal lobe epilepsy (TLE) patients were compared with neurologically normal post-mortem control subjects for neuropathology and GABAs receptor density and affinity. Neuronal loss was observed in most of the hippocampal subregions, whereas in subiculum any significant difference was detected. For GABAa receptor density (Bmax) a significant reduction was reported in CA3, CA4, and DG; the affinity was increased exclusively in DG. After the correction of Bmax value for the neuronal loss a significant increase was seen in CA3. Oligonucleotides were designed to investigate the two GABAB1 isoforms (la, and lb), and the GABAa2 subunit in human hippocampal control and HSITLE tissues, obtained as well as for the autoradiography. GABAsla, GABAB1b, and GABAB2 transcript distribution was in agreement with the receptor protein localisation, even though in the human hippocampus GABAa2 has to be yet localised and to verify if it is associated with GABAsla or GABABlbto form the dimeric active receptor. The present study suggests an involvement of GABABreceptors in HS/TLE in some not all the hippocampal subregions in terms of receptor density and affinity. Further investigations in regard to the quantitative in situ hybridisation data and the immunocytochemical results are fundamental to gain insight into the pathological role of GABAs receptors.
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Hoyle, Jane Anthea. "In situ hybridisation for the detection of viral nucleic acids." Thesis, University of St Andrews, 1991. http://hdl.handle.net/10023/13924.
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The technique of in situ hybridisation was optimised for the detection of viral RNA using radioactively-labelled single-stranded DNA and RNA probes, and applied to three areas of interest. Optimum hybridisation conditions were determined in vitro using cells infected with the single-stranded negative sense RNA paramyxoviruses. Transcription of RNA probes was the most rapid and efficient method of probe labelling, since electrophoretic purification was not required and large amounts of RNA were produced. However, their use for in situ hybridisation was problematic due to RNase contamination and low sensitivity. In contrast, DNA probes produced from M13 clones and oligonucleotide probes gave consistent hybridisation results and were preferred in subsequent studies for their ease of use, stability and sensitivity. The effect of virus-host interactions on the clearance of the paramyxovirus, SV5, in a mouse model was investigated by detection of viral RNA and protein in lung sections. Immunisation with purified SV5 proteins prior to infection provided protection against infection, indicated by a reduction in the level of viral RNA and protein, due to enhanced clearance of virus by primed T cells. X-irradiation of the host prior to infection resulted in prolonged or persistent infection in which RNA was detected up to 19 days post-infection. The potential of in situ hybridisation for detection of aetiological agents was demonstrated by investigation of the presence of measles virus in two chronic human diseases. Thus, measles virus RNA was detected in brain sections from a patient with subacute sclerosing panencephalitis and in the osteoclasts of bone sections from a patient with Paget's disease of bone. In situ hybridisation was used to analyse expression of the two immediate-early genes of herpesvirus saimiri, the 52K gene and the hinG gene. Differential expression was detected by hybridisation to mRNA using oligonucleotide probes, in productively-infected cells. The 52K gene was expressed asynchronously throughout the population in agreement with immunocytochemical detection of the 52K protein. In contrast, the hinG gene was expressed synchronously, with all cells showing similar levels of hybridisation, indicating a specific control mechanism for expression of the 52K gene, which differs from that of the hinG gene in requiring or being inhibited by additional factors. This may have relevance to the mechanism of establishment of latency in this virus.
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Hauxwell, Angela Jane. "In situ hybridisation for studying embryo development in Pisum sativum L." Thesis, University of East Anglia, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.279676.
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HajMohammadi, Sassan. "Development of FISH technology in pathological tissue." Thesis, University of Southampton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284578.
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Wu, Yih-Yiing. "Response of skin to noxious stimuli : studies using in situ hybridisation." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263124.
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Birchall, Philip Simon. "Multicolour fluorescence in situ hybridisation to RNA in whole-mount Caenorhabditis elegans." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296668.
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Zheng, Yun-Ling. "Rapid prenatal diagnosis of common fetal aneuploidies by fluorescence in situ hybridisation." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318418.
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Teo, Chong Gee. "Analysis of the Epstein-Barr virus-host relationship by in situ hybridisation." Thesis, Imperial College London, 1989. http://hdl.handle.net/10044/1/47684.
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Mohaddes, Ardebili Seyed Mojtaba. "Optimisation of interphase fluorescence in situ hybridisation for detection of common aneuploidies." Thesis, Connect to e-thesis, 1996. http://theses.gla.ac.uk/692/.
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Thesis (Ph.D.) - University of Glasgow, 1996.Ph.D. thesis submitted to the Faculty of Medicine, Department of Division of Developmental Medicine, University of Glasgow, 1996. Includes bibliographical references: p. 118-132. Print version also available.
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Hammond, David William. "Analysis of non-Hodgkin's lymphoma by conventional cytogenetics and fluorescence in-situ hybridisation." Thesis, University of Sheffield, 1995. http://etheses.whiterose.ac.uk/3064/.
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Cytogenetic analysis was performed on 40 non-Hodgkin's lymphoma (NHQ node biopsies. Chromosomes X, 3 and 12 were the most frequently gained; of the much rarer monosomies, loss of chromosome 13 was most common. Structural abnormalities primarily involved chromosomes 14,1,18,6 and 17. A markedly greater number of chromosome gains were associated with low-grade disease when compared to high-grade. In order to obtain further information from the cytogenetic analysis of the NHL karyotypes, the fluorescence in-situ hybridisation (FISH) technique was applied to the series. The activation state of additional X-chromosomes was examined and evidence that more than one X-chromosome was present in the active state in 4/9 cases was obtained. Further, in an apparent case of monosomy X, a marker was identified as an abnormal X-chromosome by chromosome painting. Interphase FISH was applied to NHL cells and numerical chromosome changes were identified; this approach was also attempted on aged bone marrow smears from acute lymphocytic leukaemia patients, in order to test the utility of the technique on archival material. Dual chromosome painting was used to elucidate the origins of add(14) chromosomes in 8 of the cases. In the control and two other cases the translocated material was demonstrated to be from chromosome 18, in two cases it was from chromosome 3 and in one case them was an insertion of chromosome 11 material. it was not possible to identify the origins of the translocated material in one NHL and in the final case the apparent add(14) was demonstrated not to contain chromosome 14 material. Structural abnormalities of chromosome 6 were investigated both by chromosome painting and by hybridisation of the MYB gene. The latter, which was initially mapped to 6q23 before hybridisation to NHL cells revealed previously unsuspected rearrangements. One case contained extrachromosomal chromatin bodies that appeared to be double minute chromosomes (dmin), which FISH analysis demonstrated to be derived from the X-chromosome and contain centromere-associated DNA. The significance of these results is discussed with reference to previously published series of NHL karyotypes.
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Jaffe, Benjamin. "Genome analysis of Hordeum bulbosum L. and hybrids with H. vulgare L." Thesis, University of Reading, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302327.
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Taylor, Clare Petronella Florence. "The use of fluorescence in situ hybridisation techniques in the diagnosis and prognosis of malignancy." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297282.
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Buckle, V. J. "The application of in situ hybridisation to the genetic analysis of the X chromosome." Thesis, University of Oxford, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370315.
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Sinclair, Paul Burdwood. "A fluorescence in-situ hybridisation and molecular investigation of leukaemia associated abnormalities of chromosome 6q." Thesis, University College London (University of London), 2002. http://discovery.ucl.ac.uk/1382604/.
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Deletions of the long arm of chromosome 6 are a recognised recurrent cytogenetic abnormality associated with ALL. Balanced rearrangements of 6q are less common but occur in leukaemias of both lymphoid and myeloid origin. With the aim of identifying genes that contribute to leukaemia through loss or rearrangement the breakpoints of translocations and deletions of 6q were mapped by FISH. Comparison between the mapped deletions led to the identification of a 4.8 Mb CDR and candidate tumour suppressor gene (GluR-6). Expression of GluR-6 was demonstrated to occur in haernatologic tissues and mutation analysis was performed on leukaemic samples with clonal deletions of the region. In one of 14 cases a base pair substitution that led to a change in amino acid sequence was found. The base pair substitution was also present in the patients' remission sample but was not seen in any of 20 other normal bone marrow samples analysed. Analysis of the translocations identified a variety of breakpoints between 6q15 and 6q27, with none positioned within the CDR. In seven cases breakpoints clustered within a 14 Mb region of 6q22-q23 but different subregions were defined for five analysed in detail. Complex rearrangements in one case of ALL appeared to result in translocation of one homologue of 6q and deletion of the second. Detailed FISH analysis defined a translocation breakpoint falling between two PACs that contained exons of a known tumour suppressor gene, the IGF2-R. Southern blot analysis failed to confirm disruption of the IGF2-R, but an IGF2-R-MRP8 fusion transcript was cloned by RACE PCR from the patients' c-DNA. Involvement of MRP8 in the translocation remains in doubt because attempts to confirm the presence of the fusion transcript were unsuccessful.
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Williams, Lisa. "Fluorescence in situ hybridisation (FISH) analysis of chromosomal aberrations in gastric tissue : the involvement of Helicobacter pylori." Thesis, Swansea University, 2004. https://cronfa.swan.ac.uk/Record/cronfa43067.
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Gastric cancer is a common cause of cancer death in the UK. It most often presents in patients when the disease is advanced, and hence treatment options are limited. As such, studies on the pre-malignant stages of gastric cancer, and interest in the mechanisms of the carcinogenic process (reactive oxygen species, ROS) and the agents that may drive the carcinogenic pathway {Helicobacter pylori infection), are important, with a view to improving disease outcome. This series of experiments has firstly shown, using CBMN assay +/- kinetochore staining and interphase FISH, that ROS causes aneuploidy of chromosomes 4, 8, 20 and 17(p53) in a human cell line. Secondly, gastric cells have been collected using endoscopic cytology brush techniques, and prepared, such that interphase FISH could be performed. Again, aneuploidy of chromosomes 4, 8, 20 and 17(p53) were detected in normal gastric mucosa, gastritis and intestinal metaplasia. The level of aneuploidy detected increased as disease severity increased. Amplification of chromosome 4, amplification of chromosome 20 and deletion of chromosome 17(p53) were the more significant findings. The degree of chromosomal abnormalities detected increased further, in a stepwise manner, when gastric dysplasia and gastric adenocarinoma cells were studied. Hence, a role for these abnormalities may exist in the initiation of, and the progression to, gastric cancer. The presence of H. pylori was also determined in the gastric tissue studied using histological analysis and PCR technology. Detection rates were comparable. The more virulent strain of H. pylori, Cag A, was found to be associated with increased disease pathology and chromosomal abnormalities, yet numbers were small. The amplification of chromosome 4 in gastric tissue was again highlighted in association with H pylori infection, hence it may reflect a role for chromosome 4 in the initiation of gastric cancer.
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Shone, John William. "Development and implementation of fluorescent in situ hybridisation for the detection of human cutaneous bacterial microflora." Thesis, Institute of Cancer Research (University Of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511179.
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Turner, Gabrielle M. "Development of in situ hybridisation to examine tissue-specific expression patterns of the invertase genes in sugarcane culm." Thesis, Stellenbosch : University of Stellenbosch, 2005. http://hdl.handle.net/10019.1/16621.
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Thesis (MSc)--University of Stellenbosch, 2005.ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situ hybridisation protocols for sugarcane culm tissue, and secondly to use the developed techniques to examine the expression patterns of three invertase isoforms in sugarcane internodes of various developmental stages. Sugarcane invertases have been the focus of intense research for many years, yet almost nothing is known of their tissue-specific distribution. It was thought that by characterising their expression patterns using in situ hybridisation, more knowledge of their functions and involvement in sucrose accumulation would be gained. Although in situ hybridisation is now regularly used to study gene expression in plants, there is to date only a single publication describing its use on immature sugarcane tissue. Therefore this technique needed further development, and this was achieved by comparing different tissue preparation methods, as well as by systematically testing the various parameters pertaining to each method. The in situ hybridization technique was also developed by testing and comparing a number of key parameters. It was found that fixing whole mount tissue for 48 h preserved sugarcane tissue adequately. High hybridization temperatures and probe concentrations provided the best signal, and including pre-treatment with HCl and Pronase was essential in sensitizing the tissue to the probe. A less viscous detection buffer reduced both osmotic effects and time required for signal detection. In the second part of this study, the developed method was used to examine the expression patterns of the three invertase isoforms in young, maturing and mature internodes of sugarcane, and the results were complemented with Northern blot analysis. Transcript of all three isoforms was found to be present in the storage parenchyma and in the phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, with soluble acid invertase declining sharply and dropping below detection in maturing and mature tissue. Transcript levels of cell wall invertase and neutral invertase declined only gradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wall invertase would appear to play important roles in phloem unloading and turgor control. Neutral invertase is suggested to be involved in either sucrose cycling or maintenance of hexose pools, however the function of this enzyme remains unclear. This study has demonstrated the value of in situ hybridization, yet at the same time has shown its limitations, especially when more traditional biochemical techniques are not employed to complement the results. Although the precise functions of the invertase isoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecific promoter design and future in situ studies of sugarcane invertases
AFRIKAANSE OPSOMMING: Die doel van hierdie projek was tweeledig: eerstens om weefselvoorbereiding en in situhibridisasie- protokolle vir die stingelweefsel van suikerriet te ontwikkel; en tweedens om die ontwikkelde tegnieke te gebruik om die uitdrukkingspatrone van drie invertaseisovorme in die suikerriet-internodes van verskeie ontwikkelingstadia te ondersoek. Suikerriet-invertases is al vir jare lank die fokus van intense navorsing, maar baie min is bekend oor hulle weefselspesifieke verspreiding. Die idee was om meer kennis oor suikerriet-invertases se funksies en betrokkenheid by sukrose-akkumulasie te verkry deur in situ-hibridisasie te gebruik om hulle uitdrukkingspatrone te karakteriseer. Alhoewel in situ-hibridisasie deesdae gereeld gebruik word om geenuitdrukking in plante te bestudeer, is daar tot op datum slegs een publikasie wat die gebruik daarvan in onvolwasse suikerrietweefsel beskryf. Hierdie tegniek moes dus verder ontwikkel word, en dit is gedoen deur verskillende weefselvoorbereidingsmetodes te vergelyk en sistematies die verskillende parameters wat op elke metode van toepassing is te toets. Die in situ-hibridisasie-tegniek is ook ontwikkel deur die toetsing en vergelyking van 'n aantal sleutelparameters. Daar is gevind dat suikerrietweefsel voldoende gepreserveer word deur die intakte gemonteerde weefsel vir 48 uur te fikseer. Hoë hibridisasietemperature en hoë peilerkonsentrasies het die beste sein gegee; die insluiting van voorbehandeling met HCl en Pronase was noodsaaklik om die weefsel meer gevoelig vir die peiler te maak. Osmotiese invloede en die tyd nodig vir seindeteksie is verminder deur die viskositeit van die buffer te verminder. In die tweede deel van die studie is die ontwikkelde metode gebruik om die uitdrukkingspatrone van die drie invertase-isovorme in jong, ontwikkelende en volwasse internodes te ondersoek en die resultate is deur 'n noordelike oordraganalise gekomplementeer. Transkripte van al drie isovorme is in die stoorparenchiem en floëemweefsel gevind. Transkripvlakke van al drie isovorme het afgeneem in ontwikkelende weefsel, met oplosbare suurinvertase wat skerp afgeneem en tot onder die deteksie-limiet gedaal het in ontwikkelende en volwasse weefsel. Transkripvlakke van selwandinvertase en neutrale invertase het slegs geleidelik afgeneem en merkbare vlakke van albei was teenwoording in ontwikkelende en volwasse weefsel. Daar word voorgestel dat suurinvertase hoofsaaklik betrokke is by internodeverlenging, terwyl selwandinvertase skynbaar 'n belangrike rol in floëem-ontlading en turgor-beheer speel. Daar word voorgestel dat neutrale invertase betrokke is óf by die sukrose-sirkulering óf by die onderhoud van heksose-poele; die funksie van hierdie ensiem is egter steeds nie duidelik nie. Hierdie studie het die waarde van in situ-hibridisasie gedemonstreer maar terselfdetyd ook die beperkinge daarvan uitgewys, veral as meer tradisionele biochemiese tegnieke nie gebruik word om die resultate aan te vul nie. Alhoewel daar onsekerheid is oor die presiese funksies van die invertase-isovorme in suikerriet, het die studie die weg gebaan vir weefselspesifieke promotorontwerp en toekomstige in situ-studies van suikerrietinvertases.
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Wall, Wilson. "A study of variation in human chromosomes shown by photometry, G, C, replication banding and in situ hybridisation." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314792.
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Stindl, Martin Maria Matthias. "Evaluation of ligation methods and the synthesis of a specific PNA-encoded peptide library." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17927.
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Dysfunctional or over and under expressed enzymes play a crucial role in a variety of diseases. A tool that can identify dis-regulated enzymes in individual patients would be beneficial and would allow personalised treatment. For this purpose, a 10,000 membered ‘spit-and-mix’ PNA-encoded peptide library with a cell penetrating peptide was synthesised and interrogated with K562 cell lysate and intact K562 cells. This allowed the specific enzyme activity pattern for ABL tyrosine kinase from both inside a cell and a lysate to be obtained. Hybridisation of this library with a DNA-microarray resulted in bio-fouling by the cell lysate, thereby preventing analysis of the phosphorylation pattern. To allow extraction and purification of the peptide library from the cell lysates, a His-tag was incorporated into the library, and enabled successful library analysis. In addition to this 10,000 member library, a focused 100 PNA-encoded peptide library was synthesised. The library included peptide sequences known to be phosphorylated by specific tyrosine kinases deregulated in acute lymphoblastic leukaemia (ALL) with a PNA-tag complementary to a DNA microarray. Different ligation methods to conjugate the peptides to PNA-tags were screened – this included amide coupling, copper catalysed azide–alkyne cycloaddition, strain promoted azide–alkyne cycloaddition and Diels–Alder cycloaddition. The inverse electron demand Diels–Alder cycloaddition between a tetrazine and norbornene was chosen as the preferred ligation method, and the reaction conditions optimised. To purify the library from cell lysate, a His-tag was again coupled to each member using the strain promoted azide–alkyne cycloaddition. To test the tetrazine ligation, fluorescence in situ hybridisation (FISH) was used in cells, whereby a fluorophore was ligated onto a tetrazine–conjugated PNA probe. This was hybridised onto an mRNA in fixed cells. Results indicated that the ligation needed further optimisation.
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Virgo, Lisa. "A study of neurotransmitter function in motor neurone disease : using the techniques of in situ hybridisation and receptor autoradiography." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336581.
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Achuthan, Rajgopal. "An evaluation of the chromosome 1p region and the BCL10 gene in malignant lymphoma, by fluorescent in-situ hybridisation." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414499.
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Ramoutar, Rakeshnie. "The development of an in situ hybridisation technique to determine the gene expression patterns of UDP-Glucose dehydrogenase, pyrophosphate-dependent phosphofructokinase and UDP-Glucose pyrophosphorylase in sugarcane internodal tissues." Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/49795.
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Thesis (MSc)--University of Stellenbosch, 2003.ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and accumulation in sugarcane is poorly understood. The present study was therefore aimed at the development of an in situ hybridisation (ISH) technique to study differential gene expression among the various cell types of the sugarcane culm. This technique in conjunction with northern and western blotting was then used to determine the sites of cellular and tissue specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose metabolism. This study revealed that the determination of the influencing parameters associated with the development of an ISH protocol was essential for the successful detection of the endogenous RNA sequences in sugarcane internodal tissues. The parameters that were investigated included the type of embedding medium, duration of fixation period, pre-treatment procedures and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the analysis of cytological gene expression at all stages of sugarcane development. The second part of this study revealed very localised transcript expression for UDP-Glc DH, PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all expressed in the parenchyma cells that were associated with the vascular bundles and the stem storage compartment, suggesting that the parenchyma cells distributed throughout the stem in the different tissue types complement each other in function for the purposes of phloem loading, unloading and assimilate transport processes. Complimentary northern and western hybridisations demonstrated that internode 7 represents a shift in the sink from utilisation to storage. This is evident by the observed decline in both the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage of development. The relative mRNA and protein abundances for the three enzymes showed a similar trend. Higher levels of the gene transcripts and translated products were observed in the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was expressed exclusively in parenchyma cells that were associated with the vascular bundles and those serving a storage function in the stem pith and UGPase was found to be localised in the phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such findings have demonstrated an increase in resolution with which gene expression can be examined at a cellular level. Hence, the results from this study have demonstrated that the knowledge of metabolic compartmentation between different tissue and cell types is a requisite to understanding the function(s) of individual enzymes within complex structures such as the sugarcane culm.
AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by sukrosemetabolisme, te bepaal. Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes. Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens alle fases van suikkerrietontwikkeling moontlik gemaak het. Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed. RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die suikerrietstingel te bepaal.
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Pedras, Maria Inês Machado. "Investigation of the regulation mechanisms for bioplastics production from industrial residues." Master's thesis, Faculdade de Ciências e Tecnologia, 2013. http://hdl.handle.net/10362/10863.
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Dissertação para obtenção do Grau de Mestre em
BiotecnologiaThe current high demand for plastics has become unsustainable. Polyhydroxyalkanoates are biopolymers stored by bacteria that can potentially replace modern plastics due to: wide range of applications; biodegradability; use of renewable resources as feedstock. High costs of current Polyhydroxyalkanoates production can be reduced using mixed cultures of organisms. Activated sludge from wastewater treatment plants is selected for Polyhydroxyalkanoates production through the imposition of cycles of intermittent feeding. In this study, the acclimation of activated sludge using synthetic volatile fatty acids (VFAs) as substrate resulted in a culture rich in Paracoccus spp. and unidentified filamentous bacteria. Low cost substrates such as sugarcane molasses (SM) or cheese whey (CW) can be employed as feedstock for further cost reduction. This requires an additional step before the microbial selection to ferment the feedstock into VFAs. In this work, the feedstock was changed from SM to CW. The population fed with SM was rich in Actinomycetaceae, while the population fed with CW was rich in Streptococcaceae, affecting the VFA composition. Consequently, the PHA-storing population and the polymer were affected. In the fermented SM (fSM) phase, the population was rich in Azoarcus (41.5 - 64.6%) and in the fCW phase the population was more diverse. Changing the pH in the fermentation reactor also affected the selection stage with an increase in Thauera and Azoarcus and a decrease in Paracoccus. A significant unidentified population of one layer sheet- forming bacteria was observed. Lastly, the occurrence of cell-to-cell communication (QS) in the selection stage was investigated. Possibly, QS molecules were detected when the carbon source was depleted. All steps of polyhydroxyalkanoate production are interconnected and for optimization, all stages must be studied and improved. Moreover, if QS proves to be involved in polyhydroxyalkanoate storage, the addition of QS molecules to the process may be explored for further optimization.
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com, dbearham@hotmail, and Douglas Bearham. "Identification and characterisation of two haplosporidian parasites of oysters in north Western Australia." Murdoch University, 2008. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20081114.120135.
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A cryptic haplosporidian parasite was detected infecting rock oysters from the Montebello Islands in north-western Australia using a PCR targeting the parasites small ribosomal subunit gene. The PCR products were cloned and sequenced along with the remaining sections of the parasites SSU rRNA gene. Phylogenetic analysis of the sequence generated indicated a Minchinia species (Haplosporidia). The SSU sequence generated was used to develop two in situ hybridisation assays to visualise the parasite in H/E sections as well as a PCR assay to detect the parasite. The molecular assays were assessed for specificity and sensitivity and were then used to compare the parasite to previous haplosporidian parasite infections of pearl oysters. Both assays produced positive results from the infected pearl oysters but not from other closely related haplosporidian species. An SEM and TEM electron microscopy analysis was performed on spores from both parasite species. The spores of the pearl oyster parasite had two spore wall filaments wound around the spore originating for a posterior thickening while the spores of the rock oyster parasite were covered in microtubule-like structures. These data suggests pearl oysters where co-infected with both the Haplosporidium sp. and the Minchinia sp. detected in rock oysters. No evidence of a posterior thickening could be found on the spores of the rock oyster parasite. Attempts to detect the parasite at the previous geographic sites of its detection in pearl oysters resulted in detection of the Minchinia species in tropical oysters in the Kimberley region of Western Australia by in-situ hybridisation.
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Bardin, Philip Greyling. "Human rhinovirus : development of an experimental disease program and detection in tissue employing in situ hybridisation and a polymerase chain reaction." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296349.
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Tholouli, Eleni. "The development of a novel quantum dot labelled oligonucleotide in situ hybridisation technique for analysis of gene expression in acute myeloid leukaemia." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.527409.
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Wilkins, Bridget Sally. "Cell-stroma interactions in haemopoiesis studied by immunocytochemistry and in situ hybridisation in long-term cultures and trephine biopsies of human bone marrow." Thesis, University of Southampton, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242221.
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Leversha, Margaret Anne. "Cytological estimations of molecular genetic difference : applications and implications of fluorescence in situ hybridisation mapping in the long arm of human chromosome 9." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337902.
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Vontell, Regina Theresa. "Expression of toll-like receptor 3 in the preterm brain after white matter injury : a post-mortem study applying immunohistochemistry and in situ hybridisation." Thesis, King's College London (University of London), 2015. https://kclpure.kcl.ac.uk/portal/en/theses/expression-of-tolllike-receptor-3-in-the-preterm-brain-after-white-matter-injury(0eaf3659-2e87-46cb-bc4d-edff67b81e4d).html.
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Toll-like receptor-3 (TLR3) has been identified in a variety of intracellular structures of cells and has been shown to detect viral molecular patterns and damage-associated molecular patterns (DAMPs). This study investigates the cellular and subcellular localization of TLR3 and its downstream signaling pathway in post-mortem brain sections from preterm infants (26-32 weeks; gestational age) with and without white matter injury (WMI) pathologies. These results were compared with a sheep model of fetal inflammation by the administration of intravenous bolus infusion of either Escherichia coli lipopolysaccharide (LPS) or saline at 102.5 +/- 0.5 days of gestation, analyzed 10 days post insult. In this study, we utilised double-labeling immunofluorescence techniques and in situ hybridization to identify the protein and messenger ribonucleic acid (mRNA) expression of TLR3. Furthermore, we assessed astroglia, microglia, oligodendroglia, neuronal populations and growth cone structures in the periventricular white matter, cortex, thalamus and in the posterior internal capsule. Additionally, we explored the TLR3 colocalisation with endosomal and phagosomal compartments. We hypothesise that after WMI or LPS exposure has occurred, the localization and activation of TLR3 alters in grey matter structures. We observed an increase in TLR3 mRNA expression in the WMI cases and the LPS exposed sheep compared to the controls. Moreover, mRNA translated into TLR3 protein expression that was more diffuse in WMI cases. We confirmed that in the WMI cases, there was an increase in TLR3 colocalisation with the endoplasmic reticulum and with the autophagosome suggesting that autophagy may be a response to stress associated with WMI. Furthermore, we show that growth cone structures and neuronal densities are altered in WMI cases. Identifying changes in TLR3 expression during fetal development may improve our understanding of disturbances seen in neuronal migration and reduced volumes of grey matter seen in preterm infants.
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Somasekar, Amudha. "Fluorescence in situ hybridisation (FISH) analysis of chromosome 1 and gene expression levels of MAD2 and BUB1 levels in premalignant stages of gastric tissue." Thesis, Swansea University, 2011. https://cronfa.swan.ac.uk/Record/cronfa43141.
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Gastric cancer is a common cause of cancer death in the world. The mortality rate from gastric cancer is high in UK as it often presents late, often with local or distant metastasis. This makes the treatment options limited. The pathogenesis of gastric cancer occurs in a multi step pathway with pre cancerous conditions leading to cancer eventually. It is important to understand this carcinogenic process (aneuploidy and abnormal gene expression levels) and the driving forces (eg. Helicobacter pylori infection) which will enable us to alter the disease outcome. This series of experiments included cytogenetic investigation which involved obtaining gastric cells using brush cytology and using Fluorescent in situ hybridisation technique to look for aneuploidy levels of chromosome 1 and 4. These two chromosomes were chosen as chromosome 1 has been recently shown to be abnormal in early in premalignant stages of gastric cancer. Chromosome 4 was chosen as hyperploidy of chromosome 4 was the predominant chromosomal aberration in Barrett’s oesophagus. This study has shown that the aneuploidy level of chromosome 1 progressively increased with the progression of the histological stages according to the Correa’s premalignant gastric cancer pathway. Significant increase in aneuploidy levels of chromosome 1 was seen in H. Pylori associated gastritis, implying that H. Pylori play a very important role in the progression of the disease. Aneuploidy can occur due to various genetic defects that may potentially occur during mitosis. Spindle cell check points play a vital role in preventing the cells from proceeding to the anaphase stage if there is any defect in the kinetochore attachment. Certain genes like MAD2 and BUB1 are thought to be instrumental in controlling the spindle cell check points and it is believed a steady state of genes like MAD2 and BUB1 are required for this. In the second part of this study, the MAD2 and BUB1 expression levels were measured and correlated to the aneuploidy stages. There was no significant difference in their expression levels in patients with significant aneuploidy level. MAD2 levels were increased in H.Pyloriassociated gastritis, which implies that H. Pylori plays an important role in the pathogenesis of gastric cancer.
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Päällysaho, S. (Seliina). "Contribution of X chromosomal and autosomal genes to species differences in male courtship songs of the Drosophila virilis group species." Doctoral thesis, University of Oulu, 2001. http://urn.fi/urn:isbn:9514265831.
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Abstract
In sympatric Drosophila species, songs produced by male wing
vibration during courtship are an effective mechanism preventing interspecific
matings and maintaining sexual isolation between different species. These songs
can vary greatly even between closely related species. The aim of this study was
to localise X chromosomal and autosomal genes affecting species differences in
male courtship song and to study their interaction in the D.
virilis group species. Various genes were probed by in
situ hybridisation on the X chromosomes of six species of the group,
which enabled us to use localised RFLP markers in QTL studies, as well as to
compare gene arrangements of different species.
Genetic analyses of differences between the songs of D.
virilis and D. littoralis showed that
species-specific song traits are affected both by X chromosomal and autosomal
genes. The X chromosomal gene(s) having a major impact on pause and pulse length
in male song were found to be located at the proximal region of the chromosome.
Precise localisation of the song genes was, however, not possible due to multiple
chromosome rearrangements restricting recombination between RFLP markers located
on this area. The same problem was faced when studying hybrids between
D. flavomontana and D. montana with
less diverged X chromosomal gene arrangements.
Interaction between the X chromosomal and autosomal song genes in determining
male song traits was studied in four species belonging to the
virilis and montana phylads of
D. virilis group. The long pauses in courtship song were
found to be mainly caused by X chromosomal song genes (or maternal / cytological
factors), while pulse length was determined by X chromosomal genes interacting
with autosomal genes. This confirms the important role of X chromosomal gene(s)
in song evolution in the montana phylad species. The
direction of dominance in hybrid songs suggests that the songs of the
montana phylad species have been affected by directional
selection favouring shorter pulses and longer pauses between sound pulses during
their evolution.
The levels and patterns of DNA polymorphism in an X-linked
fused (fu) gene was studied in
different D. montana populations. These studies revealed
that D. montana populations are significantly but not
completely isolated, and that a selective sweep at fu (or at
a gene linked to fu) may be the reason for the reduced
levels and patterns of variability of this gene in Finnish D.
montana populations. The methods used in this study will be utilized
to study variation in 'song genes' in the future.
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Caburet, Sandrine. "Structure mosai͏̈que et instabilité de l'ADN ribosomal humain : implications dans la sénescence et la cancérogenèse." Paris 7, 2002. http://www.theses.fr/2002PA077038.
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Varela, Filipa Alexandra Pereira Rosa. "Tipificação de Mycoplasma mycoides subsp. mycoides SC e detecção da sua aderência a células epiteliais pulmonares." Master's thesis, Faculdade de Ciências e Tecnologia, 2010. http://hdl.handle.net/10362/5092.
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Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaA Peripneumonia Contagiosa Bovina (PPCB) é uma doença respiratória infecciosa, de grande relevo no âmbito da produção de gado bovino, causada pela bactéria Mycoplasma mycoides subsp. mycoides SC (MmmSC). Actualmente, a PPCB permanece endémica apenas em África, sendo aí responsável por perdas incontornáveis no sector pecuário. O último surto de PPCB detectado na Europa ocorreu em Portugal em 1999 mas, devido à sua natureza insidiosa, o risco de re-emergência é permanente. As estirpes de MmmSC associadas aos últimos surtos europeus de PPCB são tradicionalmente consideradas geneticamente muito homogéneas. No entanto, trabalhos recentes de tipificação molecular revelaram a existência de uma variabilidade genética intraspecífica superior ao que se esperava. A realização deste trabalho teve como base dois objectivos fundamentais: a tipificação de estirpes de MmmSC isoladas durante os últimos surtos de PPCB que ocorreram em Portugal entre 1993 e 1998, e a aplicação de um método alternativo e inovador para a detecção e quantificação de MmmSC, após a infecção de culturas celulares. Para a concretização do primeiro objectivo foi realizada uma análise da variabilidade polimórfica de três regiões VNTR (Variable Number of Tandem Repeats) existentes no genoma de estirpes de MmmSC. O locus VNTR4 comprovou ser o mais polimórfico, detectando-se quatro perfis distintos entre as estirpes portuguesas. O perfil VNTR4 do tipo “9” (numerado em função das repetições da sequência de consenso) revelou-se amplamente distribuído geograficamente e foi predominante entre os isolados. No entanto, observou-se uma segregação geográfica de perfis, dado que na região da Beira Litoral apenas foram encontradas estirpes com o perfil VNTR4 do tipo “8”. Estes resultados sugerem que podem ter ocorrido, pelo menos, dois eventos de re-emergência de PPCB em Portugal, entre 1993 e 1998. Para a realização do segundo objectivo deste trabalho foi optimizada uma técnica baseada na hibridação in situ com sondas de DNA fluorescentes (FISH) que permitiu visualizar micoplasmas aderidos a culturas celulares. Esta técnica, que nunca tinha sido usada previamente na detecção de MmmSC, comprovou ser adequada para futuras aplicações em estudos de citoaderência e para a detecção de micoplasmas em culturas celulares.
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Karlsson, Christina. "Biomarkers in non-small cell lung carcinoma : methodological aspects and influence of gender, histology and smoking habits on estrogen receptor and epidermal growth factor family receptor signalling." Doctoral thesis, Örebro universitet, Hälsoakademin, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-19725.
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Non-small cell lung carcinoma is a leading cause of cancer mortality worldwide. There are gender and smoking associated differences both in tumour types and clinical outcome. Squamous cell carcinomas (SCC) are more frequent among smoking men while females develop adenocarcinomas (ADCA). NSCLC among never smokers are mainly ADCA, and occurs mostly in females. The present thesis elucidates the role of estrogen receptor (ER) and epidermal growth factor receptor family (EGFR/HER2-4) in NSCLC in the perspective of gender and histology as well as the influence of smoking on those biomarkers. A recently developed technique, tissue micro array (TMA), was employed.The question of how much of a tumour tissue that needed to be included in a TMA for biomarker analysis was analyzed by a statistical approach. Data indicates a sample size of three cylinders of tumour tissue with a diameter of 0.6 mm each as being appropriate and cost-effective. In order to optimally use the up to thousands of different tumour samples within a TMA, it would be optimal to serially cut and store slides before performing in situ detection of proteins and nucleic acids. Applying up to date methodology, and by evaluation with image analysis, data are presented that shows that such handling of TMA slides would be possible without any loss of biomarker information. ERα is more frequently observed in ADCA and in females and a local estradiol synthesis is supported by the presence of aromatase. ERβ is identified as a positive prognostic marker in ADCA. Smoking is associated to increased levels of ERβ mRNA. EGFR over expression is associated with a ligand. Independent phosporylation of ERα. HER-4 intracellular domain may also act as a co-activator to ERα in ADCA, especially among neversmokers. The question of ER and EGFR family signalling crosstalk as a potential target for combined targeted therapy is raised.
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Krämer, Dorothee Charlotte Agathe. "Investigation of Mammalian Chromatin Folding at Different Genomic Length Scales using High Resolution Imaging." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/19929.
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Chromatin ist ein Makromolekül, dessen Genregulation innerhalb des räumlich eingeschränkten Zellkerns organisiert werden muss. Die Genomorganisation ist eng mit Genaktivierung und Genrepression verknüpft. In den vergangenen Jahren wurde gezeigt, dass die DNA hierarchisch organisiert ist. Die Faltung läuft in aufeinander folgenden Schritten ab, wobei jede Organisationsebene sowohl zur räumlichen Komprimierung, als auch zur Genregulation beiträgt. In dieser Dissertation wurden mit Hilfe von hochauflösender Mikroskopie verschiedene Ebenen der 3D Chromatinorganisation auf Einzelzell-Basis untersucht. Auf der kleinsten Organisationsebene wurde die Struktur zweier, nebeneinander liegender topologischer Domänen (TADs) am Sox9-Lokus erforscht. Mit Hilfe von Fluoreszenz in situ Hybridisierung (FISH) in 3D Zellen, sowie Cryoschnitten in embryonalen Stammzellen von Mäusen konnten Interaktionen zwischen den benachbarten TADs festgestellt werden. FISH in Zellen mit genomischen Duplikationen, zeigte das Entstehen von zwei unterschiedlichen, durch die Duplikation entstandenen, Konformationen. Unter Verwendung von FISH wurden long-range Kontakte, die zuvor mit GAM entdeckt wurden, untersucht und es zeigte sich, dass sie häufig zwischen TADs die regulatorischen Domänen enthalten auftreten. Zudem zeigte sich die Bildung von Clustern zwischen mehreren, weit auseinander liegenden, regulatorischen Elementen. Dies lässt unter Umständen auf das Entstehen von regulatorischen Zentren zwischen diesen Enhancer-reichen Regionen schließen. Weitere Untersuchungen zeigten Veränderung der sogenannten Super-Enhancer Cluster in unterschiedlichen Zelltypen. Des Weiteren sind Super-Enhancer TADs sehr dekondensiert und wurden häufig an Splicing-Speckle Regionen vorgefunden.Chromatin needs to organize gene regulation whilst fitting into the confined space of the nucleus. Chromatin organization is therefore intertwined with gene activation and silencing. In recent years many advances in the field of chromatin architecture have been made showing that chromatin is organized hierarchically. Folding occurs in subsequent units, where each level of organization contributes to the spatial compaction of DNA and gene regulation. In this dissertation different levels of 3D chromatin organization were analysed using single-cell, high-resolution imaging. On the smallest scale, the 3D organization of two neighbouring Topologically Associating Domains (TADs) at the Sox9 locus was investigated. Performing Fluorescence in situ Hybridization (FISH) in 3D and cryosectioned mouse embryonic stem cells, extensive contacts between the two neighbouring TADs across the TAD boundary were detected. Applying FISH in a cell line bearing a genomic duplication within the Sox9 locus, the occurrence of two different conformations that result from the duplication was shown. Recent evidence from GAM showed the formation of long-range, multimer contacts between distal regulatory elements. Investigating the occurrence of long-range contacts between super-enhancer TADs in single cells by FISH, showed that they establish frequent interactions at close spatial distances. Furthermore the formation of clusters containing distal super-enhancer TADs could be demonstrated, indicating the possibility of higher-order regulatory hubs between these enhancer-rich regions. Further investigation showed that super-enhancer regions form different clusters in different cell types. Finally, it was shown that super-enhancers are highly decondensed and preferentially located at splicing speckles.
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Andersson, Ann-Catrin. "Studies on Human Endogenous Retroviruses (HERVs) with Special Focus on ERV3." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2002. http://publications.uu.se/theses/91-554-5342-2/.
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Betts, Jill Frances. "D-amino acid oxidase, D-serine and the dopamine system : their interactions and implications for schizophrenia." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:de02286f-3e33-4e3e-bc5b-222b62a28ba5.
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D-amino acid oxidase (DAO) is a flavin-dependent enzyme that is expressed in the mammalian brain. It is the metabolising enzyme of several D-amino acids, including D serine, which is an endogenous agonist at the glycine co-agonist site of the glutamatergic NMDA receptor. As such, regulation of D serine levels in the brain by DAO may indirectly modulate the activity of NMDA receptors. The expression and activity of DAO have been reported to be increased in schizophrenia. It has been identified as a putative susceptibility gene for the disorder, and as a potential therapeutic target. This thesis explored three aspects of the interface between DAO and the DA system. First, the expression of DA was investigated in the ventral tegmental area (VTA), the source of the dopaminergic mesocortical pathway. Traditionally, DAO was considered to be an enzyme confined to the hindbrain and to glia, but more recent studies have reported its expression in additional brain regions, and also in neurons. DAO mRNA and protein was found to be expressed in the VTA, and was present in both neurons and glia in this region, whereas in the cerebellum, DAO expression appeared solely glial. DA output from the VTA is regulated by NMDA receptors, and hence expression of DAO in the VTA suggests that it may serve a role in modulating cortical DA via regulation of D serine levels and NMDA receptor function. The second part of this thesis investigated the effects of DAO inhibition and D serine administration on DA levels in the prefrontal cortex (PFC) using in vivo microdialysis. Systemic DAO inhibition and D serine administration resulted in increases in extracellular levels of DA metabolites in the PFC, despite no detectable change in DA. Similarly, DA metabolites in the PFC increased after local application of D serine to the VTA, but no change was detected in DA. However, local DAO inhibition in the VTA resulted in increased levels of both DA and its metabolites, and DAO inhibition combined with D serine administration also produced increases in DA. This suggested that DAO and its regulation of D-serine levels may serve to indirectly modulate mesocortical DA function, and this may be mediated via the VTA. This notion was supported in the final section of this thesis, in which the expression of three DA genes was measured in the PFC of a novel line of DAO knockout mice. In this pilot study, there was evidence for an increase in Comt and Drd2 mRNAs in the knockout mice. As such, constitutive abolition of DAO activity may also alter mesocortical DA function. These studies provide new insights into the presence and role of DAO beyond the hindbrain, and point to a potentially important physiological function in modulating the activity of the mesocortical DA system via the VTA. This could be therapeutically relevant in the context of elevating cortical DA in the treatment of schizophrenia, and may provide supporting evidence for the clinical use of DAO inhibitors.
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Petry, Frauke. "Charakterisierung eines neuen ATP-binding-cassette-Transporters aus der ABCA-Subfamilie." Doctoral thesis, [S.l.] : [s.n.], 2004. http://webdoc.sub.gwdg.de/diss/2004/petry/petry.pdf.
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Davey, John William. "Identification of b-catenin and other RNAs in developing thalamic axons." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4011.
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This thesis provides evidence for the presence of multiple RNAs in the axons and growth cones of developing thalamic cells, particularly the mRNA for the cell adhesion and Wnt-signalling-related molecule b-catenin. After many decades of effort, mRNAs have been shown to be present in the axons of many different systems in recent years. Furthermore, these mRNAs have been shown to be locally translated at the growth cone, and this local translation is required for axons to turn in response to multiple guidance cues. As studies accumulate, it is becoming clear that different axonal systems contain different complements of mRNAs and have different requirements for local translation. One axonal system which has not been investigated to date is the thalamocortical tract. The nuclei of the thalamus are connected to the areas of the cortex via bundles of axons which travel from the thalamus to the cortex via the ventral telencephalon during embyronic development. These axons make a number of turns and are guided by many cues and other axonal tracts before innervating their cortical target. In this thesis, a quantitative real-time polymerase chain reaction (qRT-PCR) approach is developed to isolate multiple mRNAs from developing thalamic axons in vitro, including b-catenin mRNA, b-actin mRNA, 18S ribosomal RNA and ten other mRNAs. The method used should be suitable for use with other axonal systems and also for testing the effect of guidance cues on mRNA expression in axons. The qRT-PCR results for b-catenin, b-actin and 18S have been validated using in situ hybridisation. Analysis of in situ hybridisation results indicates that b-catenin and 18S, but not b-actin, are upregulated in the growth cone compared to the axon. As b-catenin has been shown to be involved in axon guidance via Slit and ephrin guidance cues in other axonal systems, and these guidance cues act upon thalamocortical axons, the identification of b-catenin mRNA in thalamic axons is an important step towards a full understanding of the thalamocortical system. The results presented here indicate that local protein synthesis is likely to occur in thalamic axons as it does in other axonal systems, and that local translation is likely to be important for thalamic axonal responses to guidance cues and other axonal tracts.
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Xu, Meng. "Specialised transcription factories." Thesis, University of Oxford, 2008. http://ora.ox.ac.uk/objects/uuid:a41d3243-c233-491a-916b-4e329cace434.
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The intimate relationship between the higher-order chromatin organisation and the regulation of gene expression is increasingly attracting attention in the scientific community. Thanks to high-resolution microscopy, genome-wide molecular biology tools (3C, ChIP-on-chip), and bioinformatics, detailed structures of chromatin loops, territories, and nuclear domains are gradually emerging. However, to fully reveal a comprehensive map of nuclear organisation, some fundamental questions remain to be answered in order to fit all the pieces of the jigsaw together. The underlying mechanisms, precisely organising the interaction of the different parts of chromatin need to be understood. Previous work in our lab hypothesised and verified the “transcription factory” model for the organisation of mammalian genomes. It is widely assumed that active polymerases track along their templates as they make RNA. However, after allowing engaged polymerases to extend their transcripts in tagged precursors (e.g., Br-U or Br-UTP), and immunolabelling the now-tagged nascent RNA, active transcription units are found to be clustered in nuclei, in small and numerous sites we call “transcription factories”. Previous work suggested the transcription machinery acts both as an enzyme as well as a molecular tie that maintains chromatin loops, and the different classes of polymerases are concentrated in their own dedicated factories. This thesis aims to further characterise transcription factories. Different genes are transcribed by different classes of RNA polymerase (i.e., I, II, or III), and the resulting transcripts are processed differently (e.g., some are capped, others spliced). Do factories specialise in transcribing particular subsets of genes? This thesis developed a method using replicating minichromosomes as probes to examine whether transcription occurs in factories, and whether factories specialise in transcribing particular sets of genes. Plasmids encoding the SV40 origin of replication are transfected into COS-7 cells, where they are assembled into minichromosomes. Using RNA fluorescence in situ hybridisation (FISH), sites where minichromosomes are transcribed are visualised as discrete foci, which specialise in transcribing different groups of genes. Polymerases I, II, and III units have their own dedicated factories, and different polymerase II promoters and the presence of an intron determine the nuclear location of transcription. Using chromosome conformation capture (3C), minichromosomes with similar promoters are found in close proximity. They are also found close to similar endogenous promoters and so are likely to share factories with them. In the second part of this thesis, I used RNA FISH to confirm results obtained by tiling microarrays. Addition of tumour necrosis factor alpha (TNF alpha) to human umbilical vein endothelial cells induces an inflammatory response and the transcription of a selected sub-set of genes. My collaborators used tiling arrays to demonstrate a wave of transcription that swept along selected long genes on stimulation. RNA FISH confirmed these results, and that long introns are co-transcriptionally spliced. Results are consistent with one polymerase being engaged on an allele at any time, and with a major checkpoint that regulates polymerase escape from the first few thousand nucleotides into the long gene.
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Bui, Loan Thuy. "Localisation of kallikreins in the prostate and association with prostate cancer progression." Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16276/1/Loan_Bui_Thesis.pdf.
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At present, prostate cancer is a significant public health issue throughout the world and is the second leading cause of cancer deaths in older men. The prostate specific antigen or PSA (which is encoded by the kallikrein 3/KLK3 gene) test is the current most valuable tool for the diagnosis and management of prostate cancer. However, it is insufficiently sensitive and specific for early diagnosis, for staging of prostate cancer or for discriminating between benign prostatic hyperplasia (BPH) and prostate cancer. Recent research has revealed another potential tumour marker, glandular kallikrein 2 (KLK2 gene/hK2 protein), which may be used alone or in conjunction with PSA to overcome some of the limitations of the PSA test. Twelve new kallikrein gene family members have been recently identified and, like hK2 and PSA, many of these genes have been suggested to be involved in carcinogenesis. In this study, the cellular localisation and level of expression of several of these newer kallikreins (KLK4, KLK5, KLK7, KLK8 and KLK11) was examined in prostate tissue, to provide an understanding of the association of their expression with prostatic diseases and their potential as additional biomarkers. Like PSA and hK2, the present observation indicated that each of these proteins, hK4, hK5, hK7, hK8 and hK11, was detected within the cytoplasm of the secretory cells of the prostate glands. For the first time, all of these newly-identified proteins were shown to be expressed in prostatic intraepithelial neoplasia (PIN) lesions, in comparison to normal glands and cancer lesions. In addition to cytoplasmic secretory cell expression, the localisation of hK4 to the basal cells and nuclei in prostatic lesions was intriguing. The intensity of hK4 staining in prostate tissue was strongest in comparison to the other newly-identified kallikrein proteins (hK5, hK7, hK8 and hK11). Therefore, KLK4/hK4 expression was characterised further to define this cellular localisation and examined in non-prostatic tissue and also in a larger number of prostate tissues in an attempt to determine its potential value as a biomarker for prostate disease. Three hK4 antipeptide polyclonal antibodies, derived against N-terminal, mid-region and C-terminal hK4 amino acid sequences, were used. The hK4 N-terminal antipeptide antibody was used to demonstrate the cellular localisation of hK4 in kidney, salivary glands, liver, testis, colon carcinoma, heart, endometrium and ovarian cancer, for the first time. The presence of hK4 in these non-prostate tissues was consistent with the previous reports using RT-PCR. The dual cytoplasmic and nuclear localisation of hK4 observed in the prostate above was also seen in these tissues. Although hK4 was found widely expressed in many human tissue types, indicating that it is not prostate specific in its expression, the highest expression level of hK4 was seen in the prostate. Therefore, detailed expression patterns and levels of KLK4 mRNA and hK4 protein in the normal prostate and prostatic diseases and histopathological lesions were investigated and reported for the first time in this study. Twelve benign prostatic hyperplasia (BPH), 19 adenocarcinoma (Gleason grade 2-5) and 34 bone metastases from prostate cancer were analysed. Using in situ hybridisation, the expression of KLK4 mRNA was detected in the cytoplasm of the secretory cells of both normal and diseased prostate tissue. KLK4 mRNA was also noted in both secretory and basal cells of PIN lesions, but the basal cells of normal glands were negative. Using the hK4 N-terminal and mid-region antipeptide antibodies, hK4 was predominantly localised in the cytoplasm of the secretory cells. The intensity of hK4 staining appeared lowest in normal and BPH, and increased in PIN lesions, high Gleason grade prostate cancer and bone metastases indicating the potential of hK4 as a histopathological marker for prostatic neoplasias. Further studies are required with a larger cohort to determine its utility as a clinical biomarker. Small foci of atypical cells, which were found within normal glands, were also intensely stained. Surprisingly, hK4 protein was found in the nucleus of the secretory cells (but not the basal cells) of high grade PIN and Gleason grade 3 prostate cancer. The detection of KLK4 mRNA and hK4 protein in PIN lesions and small foci of atypical cells suggests that up-regulation of KLK4 expression occurs early in the pathology of prostate carcinogenesis. The finding of basal cell expression is not typical for the kallikreins and it is not clear what role hK4 would play in this cell type. With the use of the hK4 C-terminal antipeptide antibody, the staining was mainly localised in the nuclei of the secretory cells of the prostate glands. Although the nuclear localisation was readily noted in more than 90% of epithelial cells of the prostate gland with the C-terminal antibody, no difference in staining intensity was observed among the histopathological lesions of the prostate. The prominent nuclear localisation with the C-terminal antipeptide antibody was also shown to be distributed throughout the nucleus by using confocal microscopy. Further, by using gold-labelled particles for electron microscopy, the intracellular localisation of these hK4 antipeptide antibodies was reported here for the first time. Similar to the immunohistochemical results, the cytoplasm was the major site of localisation with the N-terminal and mid-region antipeptide antibodies. To further characterise the involvement of KLK4/hK4 in human prostate cancer progression, the transgenic adenocarcinoma mouse prostate (TRAMP) model was used in this study. In this study, mouse KLK4 (also known as enamel matrix serine protease -1, EMSP-1) was shown to be expressed in the TRAMP prostate for the first time. Previous studies had only shown the developing tooth as a site of expression for EMSP-1. The level of EMSP-1 mRNA expression was increased in PIN and prostate cancer lesions of the TRAMP model, while negative or low levels of EMSP-1 mRNA were seen in normal glands or in control mouse prostate tissue. The normal mouse prostate did not stain with any the three hK4 antipeptide antibodies. hK4 N-terminal and mid-region antipeptide antibodies showed positive staining in the cytoplasm of the epithelial cells of PIN and cancer lesions of the mouse prostate. The C-terminal antipeptide antibody showed distinctively nuclear staining and was predominantly localised in the nuclei of the glandular cells of PIN and cancer lesions of the mouse prostate. The expression patterns of both the mRNA and protein level for mouse KLK4 strongly supported the observations of KLK4/hK4 expression in the human prostate and further support the utility of the TRAMP model. Overall, the findings in this thesis indicate a clear association of KLK4/hK4 expression with prostate cancer progression. In addition, several intriguing findings were made in terms of cellular localisation (basal as well as secretory cells; nuclear and cytoplasmic) and high expression in atypical glandular cells and PIN, perhaps indicating an early involvement in prostate disease progression and, additionally, utility as basal cell and PIN histological markers. These findings provide the basis for future studies to confirm the utility of hK4 as a biomarker for prostate cancer progression and identify functional roles in the different cellular compartments.
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Bui, Loan Thuy. "Localisation of kallikreins in the prostate and association with prostate cancer progression." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16276/.
Full textAbstract:
At present, prostate cancer is a significant public health issue throughout the world and is the second leading cause of cancer deaths in older men. The prostate specific antigen or PSA (which is encoded by the kallikrein 3/KLK3 gene) test is the current most valuable tool for the diagnosis and management of prostate cancer. However, it is insufficiently sensitive and specific for early diagnosis, for staging of prostate cancer or for discriminating between benign prostatic hyperplasia (BPH) and prostate cancer. Recent research has revealed another potential tumour marker, glandular kallikrein 2 (KLK2 gene/hK2 protein), which may be used alone or in conjunction with PSA to overcome some of the limitations of the PSA test. Twelve new kallikrein gene family members have been recently identified and, like hK2 and PSA, many of these genes have been suggested to be involved in carcinogenesis. In this study, the cellular localisation and level of expression of several of these newer kallikreins (KLK4, KLK5, KLK7, KLK8 and KLK11) was examined in prostate tissue, to provide an understanding of the association of their expression with prostatic diseases and their potential as additional biomarkers. Like PSA and hK2, the present observation indicated that each of these proteins, hK4, hK5, hK7, hK8 and hK11, was detected within the cytoplasm of the secretory cells of the prostate glands. For the first time, all of these newly-identified proteins were shown to be expressed in prostatic intraepithelial neoplasia (PIN) lesions, in comparison to normal glands and cancer lesions. In addition to cytoplasmic secretory cell expression, the localisation of hK4 to the basal cells and nuclei in prostatic lesions was intriguing. The intensity of hK4 staining in prostate tissue was strongest in comparison to the other newly-identified kallikrein proteins (hK5, hK7, hK8 and hK11). Therefore, KLK4/hK4 expression was characterised further to define this cellular localisation and examined in non-prostatic tissue and also in a larger number of prostate tissues in an attempt to determine its potential value as a biomarker for prostate disease. Three hK4 antipeptide polyclonal antibodies, derived against N-terminal, mid-region and C-terminal hK4 amino acid sequences, were used. The hK4 N-terminal antipeptide antibody was used to demonstrate the cellular localisation of hK4 in kidney, salivary glands, liver, testis, colon carcinoma, heart, endometrium and ovarian cancer, for the first time. The presence of hK4 in these non-prostate tissues was consistent with the previous reports using RT-PCR. The dual cytoplasmic and nuclear localisation of hK4 observed in the prostate above was also seen in these tissues. Although hK4 was found widely expressed in many human tissue types, indicating that it is not prostate specific in its expression, the highest expression level of hK4 was seen in the prostate. Therefore, detailed expression patterns and levels of KLK4 mRNA and hK4 protein in the normal prostate and prostatic diseases and histopathological lesions were investigated and reported for the first time in this study. Twelve benign prostatic hyperplasia (BPH), 19 adenocarcinoma (Gleason grade 2-5) and 34 bone metastases from prostate cancer were analysed. Using in situ hybridisation, the expression of KLK4 mRNA was detected in the cytoplasm of the secretory cells of both normal and diseased prostate tissue. KLK4 mRNA was also noted in both secretory and basal cells of PIN lesions, but the basal cells of normal glands were negative. Using the hK4 N-terminal and mid-region antipeptide antibodies, hK4 was predominantly localised in the cytoplasm of the secretory cells. The intensity of hK4 staining appeared lowest in normal and BPH, and increased in PIN lesions, high Gleason grade prostate cancer and bone metastases indicating the potential of hK4 as a histopathological marker for prostatic neoplasias. Further studies are required with a larger cohort to determine its utility as a clinical biomarker. Small foci of atypical cells, which were found within normal glands, were also intensely stained. Surprisingly, hK4 protein was found in the nucleus of the secretory cells (but not the basal cells) of high grade PIN and Gleason grade 3 prostate cancer. The detection of KLK4 mRNA and hK4 protein in PIN lesions and small foci of atypical cells suggests that up-regulation of KLK4 expression occurs early in the pathology of prostate carcinogenesis. The finding of basal cell expression is not typical for the kallikreins and it is not clear what role hK4 would play in this cell type. With the use of the hK4 C-terminal antipeptide antibody, the staining was mainly localised in the nuclei of the secretory cells of the prostate glands. Although the nuclear localisation was readily noted in more than 90% of epithelial cells of the prostate gland with the C-terminal antibody, no difference in staining intensity was observed among the histopathological lesions of the prostate. The prominent nuclear localisation with the C-terminal antipeptide antibody was also shown to be distributed throughout the nucleus by using confocal microscopy. Further, by using gold-labelled particles for electron microscopy, the intracellular localisation of these hK4 antipeptide antibodies was reported here for the first time. Similar to the immunohistochemical results, the cytoplasm was the major site of localisation with the N-terminal and mid-region antipeptide antibodies. To further characterise the involvement of KLK4/hK4 in human prostate cancer progression, the transgenic adenocarcinoma mouse prostate (TRAMP) model was used in this study. In this study, mouse KLK4 (also known as enamel matrix serine protease -1, EMSP-1) was shown to be expressed in the TRAMP prostate for the first time. Previous studies had only shown the developing tooth as a site of expression for EMSP-1. The level of EMSP-1 mRNA expression was increased in PIN and prostate cancer lesions of the TRAMP model, while negative or low levels of EMSP-1 mRNA were seen in normal glands or in control mouse prostate tissue. The normal mouse prostate did not stain with any the three hK4 antipeptide antibodies. hK4 N-terminal and mid-region antipeptide antibodies showed positive staining in the cytoplasm of the epithelial cells of PIN and cancer lesions of the mouse prostate. The C-terminal antipeptide antibody showed distinctively nuclear staining and was predominantly localised in the nuclei of the glandular cells of PIN and cancer lesions of the mouse prostate. The expression patterns of both the mRNA and protein level for mouse KLK4 strongly supported the observations of KLK4/hK4 expression in the human prostate and further support the utility of the TRAMP model. Overall, the findings in this thesis indicate a clear association of KLK4/hK4 expression with prostate cancer progression. In addition, several intriguing findings were made in terms of cellular localisation (basal as well as secretory cells; nuclear and cytoplasmic) and high expression in atypical glandular cells and PIN, perhaps indicating an early involvement in prostate disease progression and, additionally, utility as basal cell and PIN histological markers. These findings provide the basis for future studies to confirm the utility of hK4 as a biomarker for prostate cancer progression and identify functional roles in the different cellular compartments.
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Henwood, Anthony F. "The demonstration of estrogen receptors in various tumours a study using immunohistochemistry and in situ hybridisation /." 2004. http://hdl.handle.net/2440/37705.
Full textAbstract:
In order to study the incidence of Estrogen Receptors (ER) in breast carcinoma, lung carcinoma and melanoma, an in situ hybridisation technique for ER mRNA (ER mRNA-ISH) was developed. Various technical aspects of the procedure including tissue fixation, hybridisation conditions, and demonstration technique were investigated in order to obtain an optimum technique for routine use. ISH results were compared with ER immunohistochemistry using the monoclonal antibodies ER1D5 and D5. Commercially available biotin labelled antisense oligonucleotides to ER, Poly A (total mRNA), and sense chromogranin (negative control) were applied to frozen and formalin-fixed paraffin sections of breast carcinomas. For frozen sections, various fixatives including formalin, alcohol, Schoobridge, Zamboni's and acetic- alcohol were compared. A direct streptavidin- eroxidase and an indirect demonstration method using anti-biotin were also compared. The effect of differing formamide concentrations and post hybridisation stringency washings were analysed. An optimised ISH technique was then applied to frozen sections of 21 cases of breast carcinoma and 11 cases of lung carcinoma. Results were compared to H222 staining on adjacent sections. The ISH technique was also optimised for use on formalin-fixed, paraffin-embedded sections of 28 breast carcinomas and 17 melanomas. The results were compared with ER1D5 and D5 immunohistochemistry done on adjacent sections. The occurrence of endogenous biotin was also studied on a range of normal tissues. Consistent ISH results were obtained when formamide was omitted from the hybridisation cocktail, high stringency post hybridisation washes were discarded, room temperature hybridisations and an indirect demonstration method were used. Fixation of frozen sections in acetic/ethanol gave more consistent results with good morphology and resulted in positive nucleolar staining in 90% of breast and 45% of lung carcinomas. Positive nucleolar staining was also present in frozen sections of one metastatic melanoma. In formalin fixed paraffin sections, acid hydrolysis and pronase treatment were required prior to ISH. Cytoplasmic and/or nucleolar ER mRNA-ISH staining was seen in 87% of breast carcinoma and 97% of melanoma studied. ER1D5 was present in 54% of breast carcinomas but was absent in all melanomas. D5, on the other hand, was found in 88% of the melanomas. In conclusion, ER mRNA-ISH can be successfully done on acetic/alcohol fixed frozen sections and formalin fixed paraffin sections. Formamide, high stringency washes and elevated hybridisation temperatures are detrimental to a successful ISH reaction and an indirect demonstration method (using anti-biotin) is preferred. Unfortunately, endogenous biotin can cause false positive ISH reactions and needs to be considered during interpretation. Results show that the localisation of ER mRNA in the nucleolus is specific. Both ER mRNA-ISH and ER immunohistochemistry indicate that melanomas and some lung carcinomas contain a receptor possibly similar to that in breast carcinomas.Thesis (M.Sc.)--Department of Anatomical Sciences, 2004.
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Friedman, Brett. "Characterisation of a chromosomal translocation in an ovarian carcinoma cell line using fluorescence 'in situ' hybridisation." Thesis, 1996. http://hdl.handle.net/10539/22288.
Full textAbstract:
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Master of Science (Haematology)The region Ilpl3-pl5 on the short arm of chromosome 11 (lip) has been implicated in the initiation or progression of several human malignancies including the embryonic rhabdomyosarcoma Wilms' tumour, bladder, renal cell and ovarian carcinoma. In this study, Fluorescence In Situ hybridisation (FISH) was used to identify the nature of a chromosome llp+ abnormality present in two ovarian carcinoma cell lines after conventional cytogenetic techniques had failed to elucidate the chromosomal origin of the abnormality. Using whole chromosome library probes, the abnormality in cell line UW0V2 was found to be composed entirely of chromosome 3 material representing the translocation t (3;11) (pl2-14;pl5). In the protein-free subline UW0V2(Sf), the abnormality was found to consist of the complex translocation t (3;8; 11) (pl2-14 ;q22-24;pl5) . It is possible that the involvement of chromosome 8 in this translocation was a cell culture phenomenon. Other structural and numerical abnormalities elucidated with FISH in cell line UW0V2(Sf) included lq+, +5, +7, 7q-, 8q+, +12, +14, 14q+, -15, 16q- and -18. Using FISH together with the gene probe pSB|5 and the CEPH YAC probes 892g9, 785e5, 847al2, 954f4, 966e8 and 845a3, the breakpoint region on chromosome 11 in the _ two cell lines was narrowed down and mapped to the region Ilpl4.3-pl5.1 lying between probes 966e8 (D11S902) and 845a3 (D11S899). This represents a physical distance of approximately 1 Mb. The breakpoint in the two cell lines appeared to involve the same region on llplS.l. In a separate study, three epithelial ovarian tumour specimens and four ascitic fluid specimens were obtained. Tumour specimens T2 and T4 and ascitic fluid specimens AF-1, AF-2 and AF-3 were all cytogenetically uninformative. Cytogenetic analysis of specimen T5 revealed a single clonal abnormality involving a deletion in the region 6q21. Ascitic fluid specimen AF-5 yielded cytogenetically normal metaphases. Both specimens were hypodiploid and revealed a cytogenetically normal chromosome 11. Using FISH and CEPH YAC probes 966e8 and 845a3, no abnormalities were detected in the region llpl4.3-pl5.1 in these two specimens but one cannot rule out the possibility of submicroscopic abnormalities lying within the region between these probes. From this study we speculate that chromosome 6 abnormalities may be important in the initiation of these tumours. From the results obtained with cell lines UW0V2 and UW0V2 (Sf) we speculate that the chromosome 3 abnormalities were an early event in the evolution of these tumours while the chromosome 11 abnormality was a later event. Little is known about the region llpl4.3-pl5.1 and very few disease loci have been assigned to this region, however, we may speculate that this region harbours a tumour suppressor gene or an oncogene whose disruption or activation is critical to the pathophysiology of ovarian carcinoma and other genitourinary cancers.
WHSLYP2017
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Swaneburg, Uwe [Verfasser]. "Detektion von Porphyromonas gingivalis mittels Fluoreszenz-in-Situ-Hybridisation (FISH-Technik) / vorgelegt von Swaneburg, Uwe, geb. Patzer." 2006. http://d-nb.info/992236258/34.
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Peterková, Kristýna. "Lokalizace a kvantifikace mRNA kódující trávící peptidázy motolice Fascioloides magna." Master's thesis, 2019. http://www.nusl.cz/ntk/nusl-397285.
Full textAbstract:
Trematode peptidases are important molecules responsible for biocatalysis in many basal biological processes and are crucial in host-parasite interactions. Therefore, these enzymes are intensively studied in order to characterize their biological functions and to use them as potential diagnostic or therapeutic targets. Lately, investigation of transcriptome and secretome revealed, that adult Fascioloides magna (giant liver fluke) expresses and secretes a variety of peptidases. Thus, this thesis focuses on three newly identified enzymes: cathepsin L (FmCL), cathepsin B (FmCB) and cathepsin D (FmCD). In other trematode species, these cathepsins are being linked mainly with the digestion of host blood. We applied quantitative PCR (qPCR) to investigate relative expression levels of the three enzymes among three developmental stages - egg, miracidium and adult. It was revealed that all cathepsins have the highest expression in adult flukes in comparison to eggs and miracidia. We also localized the place of transcription of FmCL, FmCB and FmCD in adult fluke using RNA in situ hybridization. All of the peptidases were detected in gastrodermis, and in addition, they were localized in the reproductive system. The latter surprising finding is suggesting that these enzymes might have multiple functions in adult F....
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Wedi, Edris. "Untersuchung des CFL-Phänotyps ("congenital fused labia" ) in dem Neuweltaffen Common Marmoset (Callithrix jacchus) unter demographischen, physiologischen und zytogenetischen Gesichtspunkten." Doctoral thesis, 2010. http://hdl.handle.net/11858/00-1735-0000-0006-AFBB-D.
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Gibson, Catherine Elizabeth. "The expression of hydrolytic enzymes in germinating barley grain." Thesis, 2017. http://hdl.handle.net/2440/114266.
Full textAbstract:
Modification of the barley grain endosperm in germination is fundamental to successful plant growth but also has important ramifications for down-stream industrial uses of barley, particularly in the malting and brewing industries. There are a battery of enzymes that are involved in the modification process but the sites of synthesis and action of only of a few of these have been described in detail and most have only been studied in isolated tissues.
The development of a sensitive and robust in situ mRNA hybridization (ISH) procedure, which allows the detection of specific transcripts representing proteins in grain sections is described here. This first required the optimization of grain fixation, embedding and sectioning procedures as well as the adaptation of a staining method using calcofluor white to accurately assess the modification state of each grain prior to processing. Once these technical parameters were established, the non-radioactive ISH protocol was developed to allow the detection of transcripts of (1,3;1,4)-β-glucanases, (1→4)-β-endo-xylanases, limit dextrinase and limit dextrinase inhibitor in grain sections of the two barley cultivars Sloop and Himalaya. The panel of enzymes selected for study covers several aspects of grain modification, such as (1,3;1,4)-β-glucan, xylan and starch breakdown, which are all important for successful grain germination.
The successful ISH technique proved sensitive enough to discriminate between the (1,3;1,4)-β-glucanase and xylanase isoenzymes and clearly defined whether the transcipts for these enzymes were synthesized in both a tissue-specific manner and a fixed temporal sequence during grain germination. The use of monoclonal antibodies specific for the two (1,3;1,4)-β-glucanase isoforms in a related immunolabelling procedure, using the same fixed grains, also allowed the patterns of transcript and protein appearance to be correlated.
As expected, use of the ISH method showed that the transcripts of the (1,3;1,4)-β-glucanase, xylanase and limit dextrinase inhibitor genes are variously found in the aleurone cells, the starchy endosperm tissue and the scutellum. However, there were also substantial amounts of transcript detected in the tissues of the growing embryo which suggests that these enzymes may also contribute substantially to early seedling development.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2018
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Kotz, Matěj. "Karyotypová evoluce u vybraných čeledí entelegynních pavouků." Master's thesis, 2020. http://www.nusl.cz/ntk/nusl-435838.
Full textAbstract:
The Araneoidea superfamily is a diverse clade of spiders with a great species diversity. The whole superfamily displays considerable conservativeness of observed karyotypes. Most likely ancestral karyotype in males is 24 acrocentric chromosomes with X1X2 sex determination system. The goal of this study is to explore the karyotype diversity of two araneoid families - Araneidae and Mimetidae. The majority of studied species exhibit the ancestral karyotype. In some species of the aformentioned families was observed sudden increase in chromosome numbers, up to 2n♂ = 52 in Araneidae and up to 2n♂ = 57 in Mimetidae. The latter number is the highest chromosome count observed in Entelegynae so far. Increase in 2n goes hand in hand with increase in sex chromosome numbers, leading up to X1X2X3X40 system in Araneidae and up to X1X2X3X4X5X6X70 in Mimetidae. I suggest polyploidy as a possible mechanism of the increase. To test this hypothesis, I measured the size of the genome using flow cytometry and used fluorescence in situ hybridization for the detection of 18S rRNA and 5S rRNA genes. For one species, probe for U2 snRNA gene was also optimized as part of this thesis. In many species studied, these techniques were used for the first time ever. In the case of the family Mimetidae, the largest genomes in...